Celiac disease, also known as Celiac Sprue, is a multifactorial inflammatory disease of the small intestine that affects approximately 1% of the population. There is a genetic component to the disease, and an autoimmune component, but the triggering cause of celiac disease is the response to gluten proteins and peptides derived therein. In susceptible individuals, ingestion of gluten leads to the stimulation of T cells specific for gluten-derived peptides and the induction of an inflammatory response.
Dermatitis herpetiformis is a dermatologic disorder that is also controllable by limiting intake of gluten. Therefore, treatments for celiac disease may also be useful for treatment of dermatitis herpetiformis.
Celiac disease typically presents with diarrhea, but it also presents with failure to thrive in young children, anemia, neurological problems, and osteroporosis (Green, P H R and Jabri, B. Celiac disease. Annu. Rev. Med. 57:14.1-14.15 (2006) (pre-publication online)). Some patients have atypical presentations or are asymptomatic. However, even among asymptomatic patients, nutritional deficiencies can develop.
Relatives of celiac disease patients are at increased risk of developing celiac disease. Celiac disease is diagnosed by testing for the presence of IgA endomysial and tissue transglutaminase antibodies. A definitive diagnosis requires a biopsy of the upper small intestine. The characteristics of celiac disease include villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis. A positive clinical effect of gluten withdrawal from the diet is also used in the diagnosis of the disease.
Celiac disease can be effectively controlled by rigorously excluding gluten from the diet. However, maintaining a gluten-free diet is very difficult because of the ubiquity of gluten-containing products. Gluten is an integral component of wheat, barley and rye. Gluten is frequently present in small amounts, even in products that are not primarily grain-based. Maintaining a strict gluten-free diet is difficult and expensive. Therefore, there is a need for products that can be used to reduce the exposure of celiac disease patients to the toxic effects of gluten.
Gluten proteins are the storage proteins found in wheat, rye and barley grains. The gluten proteins are encoded by at least 100 genes (Jabri, B, Kasarda, D D, and Green, P H R. Innate and adaptive immunity: the Yin and Yang of celiac disease. Immunol. Rev., 206:219-231 (2005)).
Wheat gluten is isolated from wheat flour by working wheat flour dough under a stream of water. The starch fraction is washed away, leaving gluten. This preparation normally containing about 75% by weight protein, 8% by weight lipid, and with the remainder being ash, fiber and residual starch. Although a gluten ball cannot be washed from rye or barley flour doughs, the celiac disease community calls the proteins derived from rye and barley gluten, because they are close in amino acid sequence to gluten proteins of wheat and because they are active in celiac disease.
There are two major components of gluten, the gliadins, which are monomeric proteins, and the glutenins, which are polymeric proteins bound together by disulfide bonds.
Gliadin is a single-chained protein having an average molecular weight of about 30,000-40,000, with an isoelectric of pH 4.0-5.0. There are four classes of gliadin proteins: α-gliadin, β-gliadin, γ-gliadin, and ω-gliadin. Gliadin proteins are extremely sticky when hydrated and have little or no resistance to extension. Gliadin is responsible for giving gluten dough its characteristic cohesiveness. Glutenin is a larger, multi-chained protein with an average molecular weight of about 3,000,000 ranging from 100,000 to several million. The isoelectric pH of glutenin is about 6.5-7.0. The glutenin fraction is broken down into two main classes, the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits. Glutenin is resistant to extension and is responsible for the elasticity of gluten dough.
Generally, wheat gluten is fractionated into gliadin and glutenin proteins by initially solubilizing the gluten in dilute acid and then adding ethanol until a 70% solution is achieved. The solution is then neutralized with base and left to stand overnight at refrigeration temperatures. The ethanol-soluble gliadins go into solution while the glutenins precipitate out. Final separation involves decantation or centrifugation to yield the separate proteinaceous fractions (from U.S. Pat. No. 5,610,277). Alternatively, U.S. Pat. No. 5,610,277 describes an alcohol-free method for separating gliadin and glutenin. Therefore, the methods used to separate and define the wheat fractions of gluten, gliadin and glutenin are well understood in the art. Both wheat gluten and wheat gliadin can be purchased from SigmaAldrich.
The pathogenesis of celiac disease is fairly well understood. Gluten proteins are poorly digested by humans due to the large number of proline and glutamine residues, resulting in the presence of intact peptides in the small intestine. The peptides are deamidated by tissue transglutaminase 2, generating negatively charged peptides. The negatively charged peptides bind to HLA-DQ2 or HLA-DQ8, the HLA types most strongly associated with celiac disease, and stimulate T cells. Furthermore, the deamidated gluten peptides appear to activate the non-specific innate immune response, generating an inflammatory response. All classes of gliadin and glutenin proteins are apparently harmful to celiac disease patients (Jabri, B, Kasarda, D D, and Green, P H R. Innate and adaptive immunity: the Yin and Yang of celiac disease. Immunol. Rev. 206:219-231 (2005)). All of the gliadin and glutenin proteins contain large numbers of prolines and glutamines.
Although there are no therapies available for celiac disease, there are several therapies in early stages of development. These therapies are based on what is known about the pathogenesis of celiac disease.
Inhibitors of tissue transglutaminase 2 have been proposed as therapies for diseases related to gluten intolerance (see US Patent Publications 20040167069 and 20060035838). It is believed that these inhibitors would block the ability of the enzyme to deamidate glutamine residues in gluten or gluten-derived peptides, thus blocking the resulting immune activation. However, animals in which transglutaminase 2 has been selectively inactivated display impaired glucose-stimulated insulin secretion (Bernassola, F, et al., Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J. 16: 1371-1378 (2002)). Therefore, drugs inactivating this enzyme may be found to have serious health consequences and it is desirable to find more benign means of treating celiac disease.
A second approach that is being considered to treat celiac disease is to use a peptide or peptidomimetic that will bind to HLA-DQ and prevent the binding of the gluten-derived peptide and thus the resulting activation of the gluten-specific T cell response (see US patent application 20050256054). However, this approach, if successful, might be expected to have more general immunosuppressive activity. Each individual has a limited number of HLA molecules that are used to present the universe of peptides to the individual's T cells. It is possible that the dominant T cell response to a pathogen in a given individual will be restricted by the HLA-DQ allele blocked by the inhibitory peptide, thus inhibiting the protective immune response. Therefore, it is desirable to find a therapy for celiac disease that does not create the possibility of untoward immunosuppression.
A third approach that is being considered is to use an enzyme or mixture of enzymes to break down gluten in the digestive tract. The enzyme that is most widely discussed is prolyl endopeptidase, but other enzymes have been considered as well (see US patent application 20030215438). Although enzymes have been shown to degrade gluten in a variety of settings, doubts have been raised about the potential effectiveness of the enzyme in vivo. In particular, it has been suggested that the kinetics of degradation may not be sufficient to be effective in vivo, and that the enzyme will not be active as it moves through the acidic environment of the stomach (Matysiak-Budnik, T, et al. Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease. Gastroenterology, 129:786-96 (2005)). Furthermore, the activity of the enzyme may be affected by the pH of the food in which the gluten is ingested. Therefore, there is a need for a therapy that will be more predictably effective in blocking the toxicity of gluten and gluten-derived peptides.